The ruminant parasite Haemonchus contortus expresses an α1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize α1,3-fucosylated GlcNAc and terminal β-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal β-Gal, and the glycoconjugates lack the Lewis x (Lex) antigen or other related fucose-containing antigens, such as sialylated Lex, Lea, Leb Ley, or H-type 1. Direct assays of parasite extracts demonstrate the presence of an α1,3-fucosyltransferase (α1,3FT) and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT), but not β1,4-galactosyltransferase. The H. contortus α1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galα1→4GlcNAcβ1→3Galβ1→4Glc to form lacto-N-fucopentaose III Galβ1→ 4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glc, which contains the Lex antigen, and the acceptor lacdiNAc (LDN) GalNAcβ1→4GlcNAc to form GalNAcβ1→4[Fucα1 →3]GlcNAc. The α1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 μ M and a Vmax of 10.8 nmol-mg?1 h?1. The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The α1,3FT acts best with type-2 glycan acceptors (Galβ1→4GlcNAcβ1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus α1,3FT can synthesize the Lex antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.     

Pigment composition of putatively achlorophyllous angiosperms

Chlorophyll and carotenoid pigment composition was determined for ten species of putatively achlorophyllous angiosperms using high-performance liquid chromatography. Four families were represented:Lennoaceae (Pholisma arenarium);Monotropaceae (Allotropa virgata, Monotropa uniflora, Pterospora andromedea, Sarcodes sanguinea); Orobanchaceae (Epifagus virginiana, Orobanche cooperi, O. uniflora);Orchidaceae (Cephalanthera austinae, Corallorhiza maculata). Chlorophylla was detected in all taxa, but chlorophyllb was only detected inCorallorhiza maculata. The relative amount of chlorophyll and chlorophyll-related pigments in these plants is greatly reduced compared to fully autotrophic angiosperms.     

Identifying potential need for cancer palliation in Nova Scotia

OBJECTIVE: To assess the degree to which Nova Scotia cancer patients who may need palliative care are being referred to the comprehensive Halifax-based Palliative Care Program (PCP). METHODS: The authors conducted a retrospective, population-based study using administrative health data for all adults in Nova Scotia who died of cancer from 1988 to 1994. Proportions and odds ratios (ORs) were used to determine where there were differences in age, sex, place of residence, cancer cause of death, year of death and use of palliative radiotherapy between those who were referred to the PCP at the Halifax Infirmary and those who were not, and between those who were referred late (within 14 days of death) and those who were referred earlier. RESULTS: Of the 14,494 adults who died of cancer during the study period, 2057 (14.2%) were registered in the PCP. Within Halifax County, 1582 (36.4%) of the 4340 patients with terminal cancer were seen in the PCP. Predictors of PCP registration were residence in Halifax County (OR 19.2, 95% confidence interval [CI] 15.4-23.9), younger age compared with those 85 years of age or older (for those 20-54 years of age, OR 4.9, 95% CI 3.2-7.6; 55-64 years, OR 3.4, 95% CI 2.2-5.1; 65-74 years, OR 3.1, 95% CI 2.1-4.5; 75-84 years, OR 2.1, 95% CI 1.4-3.1), and having received palliative radiation (OR 1.8, 95% CI 1.5-2.2). PCP referral was associated directly with head and neck cancer (OR 5.4, 95% CI 3.0-9.7) and inversely with hematopoietic (OR 0.2, 95% CI 0.4-0.9), lymph node (OR 0.3, 95% CI 0.1-0.4) and lung (OR 0.6, 95% CI 0.4-0.9) cancer. Predictors of late referral (being referred to the PCP within 14 days of death) were age 65-84 years (OR 1.4, 95% CI 1.1-1.8) and 85 years and over (OR 1.8, 95% CI 1.1-3.0), no palliative radiation (OR 2.0, 95% CI 1.4-3.1) and cancer cause of death. People dying within 6 months of diagnosis were somewhat less likely to have been referred to the PCP (OR 0.8, 95% CI 0.6-0.9), but those who were referred were more likely to have been referred late (OR 2.6, 95% CI 2.0-3.5). INTERPRETATION: Referral to the PCP and earlier rather than late referral were more likely for younger people with terminal cancer, those who received palliative radiation and those living closer to the PCP. Referral rates also varied by cancer cause of death and the time between diagnosis and death.     

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.     

Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.     

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands—typically camelid antibodies—that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (K_D ~ 10⁻⁵–10⁻⁶ M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC_10% > 10¹³ vp/mL of resin) and product yields (~50%–80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%–80%), 80- to 400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Slipped-strand mispairing in a plastid gene: rpoC2 in grasses (Poaceae)

An exception to the generally conservative nature of plastid gene evolution is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous work by others has shown that maize and rice have an insertion in the coding region of rpoC2, relative to spinach and tobacco. To assess the distribution of this extra coding sequence, we surveyed a broad phylogenetic sample comprising 55 species from 17 angiosperm families by using Southern hybridization. The extra coding sequence is restricted to the grasses (Poaceae). DNA sequence analysis of 11 species from all five subfamilies within the grass family demonstrates that the extra sequence in the coding region of rpoC2 is a repetitive array that exhibits more than a twofold increase in nucleotide substitution, as well as a large number of insertion/deletion events, relative to the adjacent flanking sequences. The structure of the array suggests that slipped-strand mispairing causes the repeated motifs and adds to the mechanisms through which the coding sequence of plastid genes are known to evolve. Phylogenetic analyses based on the sequence data from grass species support several relationships previously suggested by morphological work, but they are ambiguous about broad relationships within the family.